应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌

目的建立敏感快速的检测猪肉中单核细胞增生李斯特菌的实时PCR方法。方法以hlyA基因为靶标,建立并验证实时PCR法的特异性。选用单核细胞增生李斯特菌CMCC 54004,制备不同浓度的纯菌液,用实时PCR进行检测,制作标准曲线并计算扩增效率。进行人工染菌实验,染菌量分别为每25g猪肉样本1.3×100、1.3×101、1.3×102、1.3×103、1.3×104、1.3×105和1.3×106CFU。分别在增菌0、4、8、12、18、24、30、36和46 h取1 ml培养液,提取DNA进行实时PCR检测,并用PCR和传统方法进行检测,比较3种方法检测的敏感性和特异性。采集24份市售猪肉样本,用这3种方法进行检测,进一步比较三者的阳性检出率。结果建立的实时PCR法特异性好,对纯菌液的检出限为1.3×103CFU/ml。人工染菌样本增菌24 h后,实时PCR检出限1.3 CFU/25 g,PCR及传统方法达到这一检出限需要增菌46 h。根据增菌24 h的检测结果,建立实时PCR样本标准曲线。24份猪肉样本,实时PCR检出17份阳性,阳性率70.83%(17/24),与PCR和传统方法的阳性率一致。根据所得的样本标准曲线,对检测样本进行定量分析,确定了阳性样本中初始含量菌。结论所建立的实时PCR具有快速简便、敏感特异等优点,整个操作可在27 h内完成,适用于猪肉中单核细胞增生李斯特菌的快速定量检测。     

单增李斯特菌不同PCR快速检测方法比较

目的比较3种PCR快速检测方法在检测单核细胞增生李斯特菌时的特异性和灵敏度方面的差异。方法针对单核细胞增生李斯特菌的毒力基因iap和prfA基因,分别设计引物,进行单个PCR、多重PCR、套式PCR等3种方法检测。结果3种PCR方法均具有较强的特异性;套式PCR的灵敏度为10²cfu/ml,高于单个PCR(10³cfu/ml)和多重PCR(10⁴cfu/ml)。结论多重PCR在特异性检测方面具有优势,而在灵敏度方面,套式PCR要明显优于单个及多重PCR方法。     

食品中产单核李斯特菌PCR检测灵敏度的研究

目的:研究PCR检测不同食品中产单核李斯特菌(Lm)的灵敏度。方法:从市场采集牛奶和火腿,向其中以及作为对照的生理盐水中添加不同量的Lm,直接检测或经过高温处理后检测Lm。结果:添加Lm的牛奶、火腿和生理盐水样品,未经高温、经100℃10 m in、经121℃15 m in处理后,PCR检测灵敏度分别为未经高温26.5、265、2.65×103cfu/m l;265、2.65×104、2.65×104cfu/m l;2.65×105、2.65×108、2.65×108cfu/m l。结论:不同食品中Lm PCR检测灵敏度不同,高温处理影响检测灵敏度。     

奶液模拟标本中单增李斯特菌real-time PCR检测方法的建立

目的建立了基于TaqMan探针的real-time PCR技术针对奶液模拟标本中单增李斯特菌的快速检测方法。方法用单增李斯特菌hlyO基因的部分片段作为靶基因,制作标准曲线,定量检测奶液中的单增李斯特菌。结果通过对不同李斯特菌及一些较为常见的致病菌的DNA进行扩增,只有单增李斯特菌能够产生扩增曲线,其余菌株均不产生扩增曲线。单增李斯特菌的检测灵敏度可以达到9copies/反应体系。结论该方法特异性好,灵敏度高,整个实验可在1.5h内完成,可用于食品中单增李斯特菌的快速检测和疫情暴发时的相关病原调查。     

实时荧光定量PCR法与常规PCR法及细菌培养法检测单增李斯特菌的比较

目的：比较实时荧光定量PCR法、常规PCR法及细菌培养法检测单核细胞增生性李斯特菌的灵敏度与特异性。方法：采用建立的实时荧光定量PCR、常规PCR及传统细菌培养法3种方法,同时对单核细胞增生性李斯特菌等细菌进行检测。结果：实时荧光定量PCR检测的灵敏度可达19 cfu/ml,且有很高的特异性,对英诺克李斯特菌等10种相关细菌均无交叉反应,从细菌核酸提取至完成检测仅需3 h左右。结论：实时荧光定量PCR检测由于在密封环境中进行,避免了产物与环境间的交叉污染,且是3种方法中最为快速敏感的方法,适用于公共卫生应急疫情的实验室快速诊断。     

生鸡肉中单核细胞增生李斯特菌的污染监测

目的：了解绍兴市部分农贸市场及超市的生鸡肉中单核细胞增生李斯特菌的污染状况。方法：监测农贸市场及超市的生鸡肉食品。结果：82份样品中检出Lm26株，总污染率为31．71％。其中农贸市场52份中检出Lm10株，总污染率为19．23％；超市30份中检出Lm16株，总污染率为53．33％。结论：大型超市生鸡肉中Lm检出率明显高于农贸市场。     

北京市售即食熟肉制品中单核细胞增生李斯特菌定量污染水平研究

目的了解北京市售即食熟肉制品中单核细胞增生李斯特菌污染情况及污染来源。方法采用MPN法定量检测即食熟肉制品中的单核细胞增生李斯特菌,在对分离菌株正确鉴定的基础上,对单核细胞增生李斯特菌分离株进行脉冲场凝胶电泳分型研究。结果 197份即食熟肉制品中有14份检出单核细胞增生李斯特菌,检出率为7.11%,平均污染水平为14.31 MPN/g。其中农贸市场采集的样品单核细胞增生李斯特菌污染率(13.89%)高于熟肉专卖店(8.57%)、超市(5.75%)和饭馆(2.56%)。而超市采集样品中该菌的平均污染水平最高,为22.78 MPN/g。共发现13种单核细胞增生李斯特菌PFGE型,其中从同一农贸市场采集的样品中分离到两株同一个PFGE型别的单核细胞增生李斯特菌,提示加工环境存在单核细胞增生李斯特菌的污染或加工过程的交叉污染。结论北京市售即食熟肉制品中存在单核细胞增生李斯特菌污染,农贸市场出售产品污染状况高于超市、熟肉专卖店和饭馆。     

改良分子信标-实时PCR快速检测产单核李斯特菌

目的：建立改良分子信标-实时PCR检测产单核李斯特菌（LMO）的快速方法，应用于食品中LMO的污染状况调查及食物中毒快速诊断。方法：根据GenBank公布的LMO hlyA基因的保守序列，设计引物和改良分子信标探针，建立改良分子信标-实时PCR检测体系，应用于食品中LMO检测。结果：改良分子信标-实时PCR反应体系DNA灵敏度为110fg，菌液灵敏度为99cfu／ml或4cfu／PCR反应体系，无交叉反应。以此反应体系检测28株LMO，均出现特异的荧光信号。上述方法可将检测时问由原来的至少4d缩短至1d。对228份食品进行LMO检测，8份增菌液LMO实时荧光PCR阳性，其中6份1310细菌培养阳性。结论：改良分子信标-实时PCR反应体系快速、灵敏度高，特异性强．能提高LMO的检出率和准确性，可应用于LMO食品污染状况调查及食物中毒的快速诊断。     

Development and evaluation of a real-time polymerase chain reaction assay targeting iap for the detection of Listeria monocytogenes in select food matrices

Listeria monocytogenes is an intracellular foodborne pathogen that has been associated with severe human illnesses. Various rapid detection methods have been developed for the specific detection of this pathogen. In the present study, a real-time quantitative polymerase chain reaction (PCR) assay targeting iap, a gene encoding extracellular protein p60, was developed for L. monocytogenes. The PCR efficiency is above 85% and the limit of detection (LOD) is 30 copies of genome per reaction for all strains tested. The assay exhibited 100% inclusivity and exclusivity rates. The detection of L. monocytogenes in five food matrices, whole milk, soft cheese, turkey deli meat, smoked salmon, and alfalfa sprouts, was evaluated with and without enrichment. Without enrichment, the LOD for all food matrices were 4×10(3)?CFU/mL food enrichment mix for whole milk and 4×10(4)?CFU/mL for all other foods. With 24?h incubation in Buffered Listeria Enrichment Broth, the LOD was 3?CFU/25?g food for whole milk, turkey deli meat, and smoked salmon and 9?CFU/25?g food for soft cheese and alfalfa sprouts. With 48?h incubation, the LOD was 3?CFU/25?g food for all matrices. This quantitative PCR appears to be a promising alternative for rapid detection of L. monocytogenes in select foods.     

食品中单增李斯特菌PCR检测方法建立与评价

目的建立单核细胞增生李斯特菌（Listeria monocytogenes，LM）快速、敏感、特异的PCR诊断方法。方法采用聚合酶链式反应技术（PCR）特异性扩增单核细胞增生李斯特菌溶血素基因（hlyA），并评价该方法的特异性与敏感性。结果在706bp处出现nuc基因的目的片断，只有单增李斯特菌的目的片段获得扩增，其他菌种扩增均呈阴性；该方法可以检测到3．3ng／L的DNA。结论PCR方法比传统细菌检测方法更特异、快速、灵敏和简便。为食品中单增李斯特菌的快速检测提供了新的手段。     

肉及肉制品中肠致病性大肠杆菌两重PCR检测方法的建立

目的　建立肉及肉制品中(EPEC)的快检方法。方法　以EPEC特异的微绒毛粘连基因(eaegene)和菌毛束形成编码基因(bfpgene)作为模板,设计两对引物对肉及肉制品中的EPEC进行特异性扩增。结果　该方法特异性好,对肉及肉制品中EPEC的最低检出量为10cfu g ,检出时间为8～17小时。结论　建立了可检测肉及肉制品中EPEC的快速、敏感、特异PCR方法。     

快速、准确的B族链球菌检测——即时荧光探针核酸扩增技术的应用

目的 研究和建立即时荧光探针核酸扩增技术,即荧光PCR检测方法,探讨B族链球菌检测的临床应用和性能.方法 首先采用标准化荧光PCR技术和操作流程,进行分析性灵敏性和特异性的鉴定,再收集临床送检样品做进一步认证.结果 分析性灵敏性达到1×102 copies/ml,分析性特异性达到特定靶序列设计要求,标准B族链球菌检测呈阳性,其他生殖道和直肠部位的易感菌株12株均阴性.并具备高重复性;以B族链球菌培养方法做对比,收集1 223例临床样本,实验结果显示:荧光PCR阳性检出率为17.58％,传统培养方法为10.87％.对PCR(+)培养(-)的72例,再扩增并测序,均检出B族链球菌的特有基因序列.结论 荧光PCR检测技术,检测B族链球菌具有较高的灵敏性和特异性,特别有助于孕妇临产前快速检测的应用.     

Evaluation of a multiplex PCR system for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 in foods and in food subjected to freezing

Conventional culture methods were compared to a multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7 from enrichment cultures of various types of artificially inoculated and naturally contaminated foods. The multiplex PCR assay was evaluated in 44 types of spiked food samples, including meat, produce, fish, and dairy products targeting genes specific for each pathogen for simultaneous detection. The sensitivity of the assay was 相似文献   

实时荧光PCR快速同时检测从业人员沙门菌和志贺菌结果分析

目的 了解双重实时荧光PCR快速同时检测从业人员沙门菌和志贺菌的效果.方法 采集从业人员肛拭子样本,用双重实时荧光PCR法检测沙门菌和志贺菌,检出的疑似阳性样本按国标法进行培养鉴定.结果 采用双重实时荧光PCR反应体系共检测从业人员39 974人次,荧光PCR检测出沙门菌阳性41份,阳性率1.22‰,培养法检测出31份,阳性率为0.78‰,沙门菌荧光PCR检测法的灵敏度为100％,特异性为99.95％.荧光PCR检测出志贺菌10份,阳性率为0.25‰,培养法检测出3份,阳性率为0.08‰,志贺菌荧光PCR法的灵敏度为100％,特异性为99.98％.从样品处理到检测结果仅需要时间2h～ld.结论 双重实时荧光PCR检测体系快速、灵敏度高、特异性强,可用于从业人员体检的快速诊断和肠道传染病的初筛,为肠道传染病的分子流行病学调查提供新的检测手段.     

Rapid detection of motile Salmonella in chicken meat by the 1-2 Test]

The 1-2 Test (BioControl) is an enrichment culture/immuno-diffusion assay for detecting Salmonella in foods by use of poly-H-antibodies. The performance of the 1-2 Test was compared with that of the traditional culture method on 85 chicken meat samples. The results were as follows: The detection frequency of Salmonella by the 1-2 Test 24-hour assay (28/85, 32.9%) was greater than that by the 8-hour assay (16/85, 18.8%) and almost equal to that by the traditional method (26/85, 30.6%). The 8-hour assay gave a 46.2% sensitivity and 93.2% specificity. The 24-hour assay gave an 84.6% sensitivity and 89.8% specificity. Detection appeared to show some influence from the number of Salmonella, number of viable cells (SPC) and coliform groups. Differences in serotypes of Salmonella detected by the 1-2 Test and the traditional methods were also observed. Although there were some differing results between the 1-2 Test and the traditional method, this system appears to be useful for detecting Salmonella in foods, since results can be obtained at least 24 hours earlier than by the conventional procedures.     

荧光PCR探针熔解曲线法检测结核分枝杆菌耐利福平突变研究

目的对荧光PCR熔解曲线法检测结核分枝杆菌耐利福平突变方法进行临床研究,评价检测能力及在国境口岸的应用价值。方法应用荧光PCR熔解曲线法检测结核病人临床分离结核分枝杆菌,以传统药物敏感性试验为标准,获得该方法的灵敏性、特异性。结果对347例结核病人临床分离培养样本,传统药物敏感性试验检出271例利福平敏感标本,76例利福平耐药标本。荧光PCR熔解曲线法检出269例利福平敏感标本,78例利福平耐药标本,灵敏性为93.42%,特异性为97.42%,符合率为96.54%。结论荧光PCR熔解曲线法检测速度快速、灵敏度高、特异性强,可用于结核分枝杆菌利福平耐药突变的快速检测,适于国境口岸对耐多药结核病的快速筛查。     

Occurrence of Campylobacter, Salmonella, Yersinia enterocolitica and Listeria monocytogenes in some retail food products in Novi Sad

The official reporting system in the Province of Vojvodina (PV) indicates that cases of human salmonellosis were partly covered by complete epidemiological investigation including laboratory analysis of the suspected food. Intestinal campylobacteriosis and yersiniosis and four cases of septicemias caused by Listeria monocytogenes were not fully epidemiologically investigated. Actual country legislation on food safety does not include provisions for a routine control of the above mentioned pathogens except for Salmonella. In the PV, there are no other sources of data that contribute to risk assessment of the above food-borne diseases. A pilot investigation, performed in Novi Sad, indicated that 8.17% out of the total number of 257 retail food samples (90 of fresh meat and 167 of ready-to-eat food) had been contaminated with one of the tested bacteria Campylobacter or Salmonella or Listeria monocytogenes. Yersinia enterocolitica was not detected in any of the tested samples. Fresh poultry meat and other fresh meats were the dominant sources of the detected pathogens compared to samples of ready-to-eat food (p < 0.05). Campylobacter was detected in 18.8% and 10.0% samples of fresh poultry and other fresh meat respectively, which was not statistically significant (p > 0.05). Salmonella was detected in 3.3% samples of fresh poultry meat. Listeria monocytogenes was detected in 5.0% samples of fresh poultry and in 3.3% samples of other fresh meat, the difference was not statistically significant (p > 0.05). One sample (0.6%) of ready to eat food was contaminated with Campylobacter and one (0.6%) with Salmonella.     

实时荧光PCR快速检测粪便中艰难梭菌方法

目的:建立实时荧光PCR快速检测艰难梭菌的方法。方法:以艰难梭菌磷酸丙糖异构酶(tpi)基因的保守序列为模板设计和合成特异性引物和荧光标记探针,建立实时荧光PCR检测体系,通过检测含有艰难梭菌标准菌株浓度为106-10 CFU/ml的细菌培养物及加标模拟样本进行敏感性分析,并对其特异性和干扰性进行评价。结果:该方法只对艰难梭菌进行特异性扩增,其他常见的病原菌均不能扩增;整个检测过程只需要2 h,对艰难梭菌菌悬液可检测至10 CFU/ml细菌,对加标粪便样本可检测至1000 CFU/ml细菌。结论:本研究建立的实时荧光PCR检测艰难梭菌方法具有快速、特异、敏感性高等优点,能实现对艰难梭菌的快速检测。     

全自动微生物磁珠分选系统检测单核细胞增生李斯特菌效果研究

目的:研究全自动微生物磁珠分选系统对食品中单增李斯特氏菌的检测效果。方法:取400份自然样品,用磁珠分选系统和国标法对样品进行对比检测。结果:全自动微生物磁珠分选系统相较国标法对自然样品中单核细胞增生李斯特氏菌阳性检出率有一定程度提高。结论:全自动微生物磁珠分选系统简单易用,标准化程度高,可以和国标检测方法相结合以提高单增李斯特氏菌的检出率。